Real-time detection of influenza virus

ABSTRACT

The present invention provides system and methods for detecting an analyte indicative of an influenza viral infection in a sample of bodily fluid. The present invention also provides for systems and method for detection a plurality of analytes, at least two of which are indicative of an influenza viral infection in a sample of bodily fluid.

CROSS-REFERENCE

This application is a divisional of U.S. patent application Ser. No.15/865,037, filed on Jan. 8, 2018, which is a continuation of U.S.patent application Ser. No. 14/155,150, filed on Jan. 14, 2014, now U.S.Pat. No. 9,885,715, which is a continuation of U.S. patent applicationSer. No. 13/187,960, filed on Jul. 21, 2011, now U.S. Pat. No.8,669,047, which is a continuation of U.S. patent application Ser. No.11/746,535, filed on May 9, 2007, now U.S. Pat. No. 8,007,999, whichclaims the benefit of U.S. Provisional Patent Application No.60/799,442, filed May 10, 2006 and U.S. Provisional Patent ApplicationNo. 60/800,939, filed May 16, 2006, each of which applications andpatents are incorporated herein by reference in their entireties for allpurposes. This application is related to application Ser. No.11/389,409, filed on Mar. 24, 2006, now U.S. Pat. No. 7,635,594, whichapplication and patent are incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

Influenza (“flu”) is an infectious disease capable of inflicting upon awide variety of hosts, including birds and mammals. Flu is caused by anRNA virus of the orthomyxoviridae family (that generally comprises thetype A, B, and C influenza viruses). Avian flu is caused by a virus ofthis family adapted to birds, thus it is also named bird flu, avianinfluenza, or bird influenza. A current pandemic threat stems from anunprecedented outbreak of the H5N1 strain of the influenza A virus inAsia and Europe. This strain has an ability to mutate and adapt itselfto a wide range of hosts, including birds and humans. The HomelandSecurity Council issued the “National Strategy for Pandemic Influenza”(“The Strategy”) in November of 2005 in response to the current pandemicthreat. A critical part of that initiative focuses on the rapididentification of Avian Flu in patients and birds. The strategy seeks toimprove the surveillance and detection of the Avian Flu.

As of November 2005, the virus causing the Avian Flu pandemic threat wasknown to have infected 121 people in four countries, resulting in 62deaths over the past two years. Those infected with H5N1 had, in almostall cases, extensive physical contact with infected birds. Although thevirus has not yet shown an ability to transmit efficiently betweenhumans, as is seen with the annual human influenza virus epidemic, itraises a serious concern that it will acquire this capability throughgenetic mutation or exchange of genetic material with a human influenzavirus.

Influenza causes approximately 36,000 deaths and more than 200,000hospitalizations each year in the U.S. alone, and costs the U.S. over$10 billion annually. In addition, the last three pandemics, in 1918,1957, and 1968, killed approximately 40 million, 2 million, and 1million people worldwide, respectively.

There remains a pressing need for devices and methods that canaccurately and rapidly detect the presence of Avian Flu to provide anearly warning of a pandemic in order to contain the spread of thedisease. An ideal system would (1) allow for retrieval, transmission,and analysis of data from such devices; and (2) provide a real-timewarning system to health and government officials. The present inventionsatisfies this need and provides related advantages.

SUMMARY OF INVENTION

The present invention provides a system for detecting an analyteindicative of an influenza viral infection in a bodily fluid from asubject. The system typically comprises a) a fluidic device, saidfluidic device comprising a sample collection unit and an assayassembly, wherein said sample collection unit allows a sample of bodilyfluid suspected to contain said analyte to react with reactantscontained within said assay assembly to yield a detectable signalindicative of the presence of said analyte; b) a reader assemblycomprising a detection assembly for detecting said detectable signal;and c) a communication assembly for transmitting said detected signal tosaid external device. The system is capable of detecting an influenzatype A, B, and/or C viral infection. In general, the analyte maycomprise a surface glycoprotein of an influenza virus, which can behemagglutinin (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12,H13, H14, H15, and H16) and/or neuraminidase (e.g., N1, N2, N3, N4, andN5). The bodily fluid can be drawn from a subject selected from thegroup consisting of human, poultry and wild birds.

The present invention also provides a system for detecting a pluralityof analytes, at least two of which are indicative of an influenza viralinfection in a bodily fluid from a subject. The system typicallycomprises a) a fluidic device, said fluidic device comprising a samplecollection unit and an assay assembly, wherein said sample collectionunit allows a sample of bodily fluid suspected to contain said pluralityof analytes to react with reactants contained within said assay assemblyto yield one or more detectable signals indicative of the presence ofsaid at least two analytes; b) a reader assembly comprising a detectionassembly for detecting said one or more detectable signals; and c) acommunication assembly for transmitting said detected signal to saidexternal device.

The present invention further provides a method of using the subjectsystems. In one aspect, the present invention provides a method fordetecting an analyte indicative of an influenza infection in a bodilyfluid of a subject. The method involves the steps of a) providing asubject system; b) allowing a sample of bodily fluid to react with thereactants contained within said assay assembly to yield a detectablesignal indicative of the presence of said analyte; and c) detecting saiddetectable signal. In another aspect, the method comprises the steps ofa) providing a fluidic device comprising at least one sample collectionunit, an immunoassay assembly containing immunoassay reagents, aplurality of channels in fluid communication with said sample collectionunit and/or said immunoassay assembly; b) actuating said fluidic deviceand directing said immunoassay reagents within said fluidic device; c)allowing a sample of bodily fluid suspected to contain said analyte toreact with said immunoassay reagents contained within said assayimmunoassay assembly to yield a detectable signal indicative of thepresence of said analyte indicative of an influenza viral infection insaid sample; and d) detecting said detectable signal generated from saidanalyte collected in said sample of bodily fluid. Where desired, thesample of bodily fluid used for such detection is less than about 500microliters. A variety of influenza viral infections can be detected.They include but are not limited to influenza type A, B, and C viralinfection.

The present invention further provides a method of detecting a pluralityof analytes, at least two of which are indicative of an influenza viralinfection in a bodily fluid from a subject. The method comprise thesteps of a) providing a fluidic device comprising at least one samplecollection unit, an immunoassay assembly containing immunoassayreagents, a plurality of channels in fluid communication with saidsample collection unit and/or said immunoassay assembly; b) actuatingsaid fluidic device and directing said immunoassay reagents within saidfluidic device; c) allowing a sample of bodily fluid suspected tocontain said plurality of analytes to react with said immunoassayreagents contained within said assay immunoassay assembly to yield oneor more detectable signals indicative of the presence of said at leasttwo analytes in said sample; and d) detecting said one or moredetectable signals generated from said plurality of analytes collectedin said sample of bodily fluid.

Also provided in the present invention is a fluidic device for detectinga type of influenza viral infection. The fluidic devices comprise acartridge comprising a plurality of reactants, at least two of which arereactive with different analytes present in a bodily fluid from asubject, wherein said different analytes are indicative of the type ofinfluenza infection. In one aspect, each of the at least two reactantsbinds to a different surface glycoprotein of an influenza virus. Thedifferent surface glycoprotein may be a member selected from the groupconsisting of hemagglutinin and neuraminidase. Any two of the followingsurface glycoproteins can be the target analytes of the at least tworeactants: hemagglutinin 1, hemagglutinin 2, hemagglutinin 3,hemagglutinin 4, hemagglutinin 5, hemagglutinin 6, hemagglutinin 7,hemagglutinin 8, hemagglutinin 9, hemagglutinin 10, hemagglutinin 11,hemagglutinin 12, hemagglutinin 13, hemagglutinin 14, hemagglutinin 15,hemagglutinin 16, neuraminidase 1, neuraminidase 2, neuraminidase 3,neuraminidase 4, and neuraminidase 5. In a preferred embodiment, one ofthe at least two reactants binds to hemagglutinin 5 and the other bindsto neuraminidase 1. Where desired, the cartridge may further comprise asample collection unit and an assay assembly. In some aspects, the assayassembly is an immunoassay assembly comprising immunoreactants.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is one embodiment showing multiple components of the presentsystem.

FIG. 2 shows different layers of an exemplary fluidic device prior toassembly.

FIGS. 3 and 4 illustrate the fluidic network within an exemplary fluidicdevice.

FIG. 5 shows a top, side, and bottom view of exemplary reagent chambersof the present invention.

FIG. 6 illustrates an exemplary side view of a reagent chamber influidic communication with a fluidic device.

FIG. 7 illustrates exemplary reagent chambers being filled withreagents.

FIGS. 8 and 9 illustrate a side view of an exemplary fluidic device iscombination with actuating elements of the reader assembly.

FIG. 10 compares a two-step assay with a competitive binding assay.

FIG. 11 shows an exemplary two-step chemiluminescence enzymeimmunoassay.

FIG. 12 shows the increased sensitivity of the two-stepchemiluminescence enzyme immunoassay.

FIG. 13 shows the ability of TOSCA to assay less than ideal samples andmaintain desired sensitivity.

FIG. 14 shows an exemplary ELISA.

FIG. 15 shows an exemplary ELISA for a virus.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention is a system for detecting an analyteindicative of an influenza viral infection present in a sample of bodilyfluid. The analyte may be indicative of an influenza type A, type B,and/or type C viral infection. The analyte may comprise at least onesurface glycoprotein of an influenza virus. Exemplary surfaceglycoproteins are, without limitation, hemagglutinins andneuraminidases. Hemagglutinin surface proteins include, but are notlimited to, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15, and H16. Non-limiting neuraminidase surface proteins include N1,N2, N3, N4, and N5. The analyte may also comprise an antibody to asurface glycoprotein of an influenza virus that is generated by theinfected host.

Another aspect of the present invention is a system for detecting aplurality of analytes, at least two of which are indicative of aninfluenza viral infection present in a sample of bodily fluid.Similarly, the analytes may be indicative of an influenza type A, typeB, and/or type C viral infection. The analytes may comprise a pluralityof surface glycoproteins of an influenza virus. In some embodiments, theplurality of surface glycoproteins comprises a hemagglutinin and aneuraminidase. The hemagglutinin may be selected from the groupconsisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13,H14, H15, and H16, and the neuraminidase may be selected from the groupconsisting of N1, N2, N3, N4, and N5. In preferred embodiments thehemagglutinin is H5 and the neuraminidase is N1. The analytes may alsobe a plurality of antibodies specific for surface glycoproteins of aninfluenza virus. The system is capable of detecting and/or quantifyingthe analytes of particular interest.

One further aspect of the present invention is system for detecting aplurality of analytes incorporated into a single entity such as a viralparticle or cell or cell fragment. In this aspect the plurality ofanalytes are preferably a combination of analytes, at least two of whichare indicative of an influenza viral infection in a sample of bodilyfluid. The analytes may be indicative of an influenza type A, type B, ortype C viral infection. The plurality of analytes may comprise acombination of surface glycoproteins of an influenza virus. In someembodiments the plurality of analytes may be a combination of surfaceglycoproteins comprising a combination of a hemagglutinin and aneuraminidase. The hemagglutinin may be selected from the groupconsisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13,H14, H15, and H16, and the neuraminidase may be selected from the groupconsisting of N1, N2, N3, N4, and N5. In preferred embodiments thecombination of analytes is associated with a virulent strain ofinfluenza such as the H5N1 combination. This aspect of the invention isspecific for detecting the combination of the plurality of analytes. Itcan distinguish between infection with a virulent strain such as acombination of H5N1 and a putative-infection with a differentcombination of analytes. One variation of this aspect of the inventionis to utilize one or more reactants reactive with one or more viralantigens (e.g., anti-viral surface glycoprotein antibody) to capture theviral particles at a reaction site, and then apply another set ofreactant (either one or multiple reactants) to specifically detect forthe bound viral particles. One exemplary set up will utilize anti-H2antibodies as the capturing antibodies, and anti-N5 antibodies,preferably enzyme-labeled anti-N5 antibodies as the detecting reagent.

In some embodiments the system detects a plurality of human antibodiesto viral antigens such as antibodies to surface glycoproteins of aninfluenza virus. These human antibodies can be circulating in theinfected subjects.

In some embodiments the analyte of interest may be a complex of ananalyte indicative of an influenza viral infection in a sample of bodilyfluid and a human antibody to the analyte. The analyte may be anyanalyte indicative of an influenza viral infection described herein, butis preferably the H5 hemagglutinin, the N1 neuraminidase, or the H5N1complex of the H5 and N1 surface glycoproteins.

A further aspect of the present invention is a system for detecting aplurality of analytes, wherein at least one analyte is indicative of aninfluenza viral infection in a sample of bodily fluid, and wherein atleast one analyte is a biomarker in the sample of bodily fluidindicative of the stress imposed on the human body by the viralinfection. The at least one analyte indicative of an influenza viralinfection may be any analyte indicative of an influenza viral infectiondescribed herein. Exemplary biomarkers indicative of the stress imposedon the human body by the viral infection include, without limitation,CRP, TNF □□□ interleukins and the like.

The subject system typically comprises a fluidic device having one ormore of the following components: a sample collection unit, an assayassembly, a reader assembly, and a communication assembly. The samplecollection unit typically allows a sample of bodily fluid collected froma subject to react with reactants contained within the assay assemblyfor generating a signal indicative of the presence of the analyte ofinterest. The reader assembly detects the signal, which is thentransmitted via the communication assembly to an external device forfurther processing.

Any bodily fluids suspected to contain an analyte of interest can beused in conjunction with the subject system or devices. Commonlyemployed bodily fluids include but are not limited to blood, plasma,blood serum, saliva, urine, gastric and digestive fluid, tears, stool,semen, vaginal fluid, interstitial fluids and cerebrospinal fluid. In apreferred embodiment, the bodily fluids are used directly for detectingthe analytes present therein with the subject fluidic device withoutfurther processing. Where desired, however, the bodily fluids can bepre-treated before performing the analysis with the subject fluidicdevices. The choice of pre-treatments will depend on the type of bodilyfluid used and/or the nature of the analyte under investigation. Forinstance, where the analyte is present at low level in a sample ofbodily fluid, the sample can be concentrated via any conventional meansto enrich the analyte. Methods of concentrating an analyte include butare not limited to drying, evaporation, centrifugation, sedimentation,precipitation, and amplification. Where the analyte is a nucleic acid,it can be extracted using various lytic enzymes or chemical solutionsaccording to the procedures set forth in Sambrook et al. (“MolecularCloning: A Laboratory Manual”), or using nucleic acid binding resinsfollowing the accompanying instructions provided by manufactures. Wherethe analyte is a molecule present on or within a cell, extraction can beperformed using lysing agents including but not limited to denaturingdetergent such as SDS or non-denaturing detergent such as Thesit®,sodium deoxycholate, Triton® X-100, and TWEEN® 20. In some embodimentssample pretreatment is accomplished automatically within the fluidicdevice.

The volume of bodily fluid to be used with a fluidic device of thepresent invention is generally less than about 500 microliters,typically between about 1 to 100 microliters. Where desired, a sample of1 to 50 microliters or 1 to 10 microliters can be used for detecting ananalyte using the subject fluidic device.

A benefit of the current invention is that only a very small volume ofblood is required to detect an analyte of interest in animals. In someembodiments between about 1 microliter and about 50 microliters aredrawn. In preferred embodiment between about 1 microliter and 10microliters are drawn. In preferred embodiments about 5 microliters ofblood are drawn from the subject.

A bodily fluid may be drawn from a subject and brought into the fluidicdevice in a variety of ways, including but not limited to, lancing,injection, or pipetting. In one embodiment, a lancet punctures the skinand draws the sample into the fluidic device using, for example,gravity, capillary action, aspiration, or vacuum force. The lancet maybe part of the fluidic device, or part of a reader assembly, or as astand alone component. Where needed, the lancet may be activated by avariety of mechanical, electrical, electromechanical, or any other knownactivation mechanism or any combination of such methods. In anotherembodiment where no active mechanism is required, a subject can simplyprovide a bodily fluid to the fluidic device, as for example, couldoccur with a saliva sample. The collected fluid can be placed in thesample collection unit within the fluidic device. In yet anotherembodiment, the fluidic device comprises at least one microneedle whichpunctures the skin. The microneedle can be used with a fluidic devicealone, or can puncture the skin after the fluidic device is insertedinto a reader assembly.

In some embodiments a microneedle is about the size of a human hair andhas an integrated microreservoir or cuvette. The microneedle maypainlessly penetrate the skin of a subject and draw a small bloodsample. More preferably, the microneedle collects about 0.01 to about 1microliter, preferably about 0.05 to about 0.5 microliters and morepreferably about 0.1 to about 0.3 microliters of capillary blood. Insome embodiments a microneedle may be constructed out of silicon and isabout 10 to about 200 microns in diameter, preferably about 50 to about150 microns in diameter, and most preferably about 100 microns indiameter, making their application to the skin virtually painless. Toensure that a capillary is actually struck by a needle, a plurality ofmicroneedles may be used for sample collection. Such microneedles may beof the type marketed by Pelikan (Palo Alto, Calif.) and/or Kumetrix(Union City, Calif.). U.S. Pat. No. 6,503,231 discloses microneedleswhich may be used with the present invention.

Microfabrication processes that may be used in making the microneedlesdisclosed herein include without limitation lithography; etchingtechniques such as wet chemical, dry, and photoresist removal; thermaloxidation of silicon; electroplating and electroless plating; diffusionprocesses such as boron, phosphorus, arsenic, and antimony diffusion;ion implantation; film deposition such as evaporation (filament,electron beam, flash, and shadowing and step coverage), sputtering,chemical vapor deposition (CVD), epitaxy (vapor phase, liquid phase, andmolecular beam), electroplating, screen printing, and lamination. Seegenerally Jaeger, Introduction to Microelectronic Fabrication(Addison-Wesley Publishing Co., Reading Mass. 1988); Runyan, et al.,Semiconductor Integrated Circuit Processing Technology (Addison-WesleyPublishing Co., Reading Mass. 1990); Proceedings of the IEEE MicroElectro Mechanical Systems Conference 1987-1998; Rai-Choudhury, ed.,Handbook of Microlithography, Micromachining & Microfabrication (SPIEOptical Engineering Press, Bellingham, Wash. 1997). Alternatively,microneedles may be molded in silicon wafers and then plated usingconventional wire cutting techniques with nickel, gold, titanium orvarious other biocompatible metals. In some embodiments microneedles canbe fashioned from biopolymers. In some embodiments microneedles may befabricated and employed for the claimed devices according to the methodsof Mukerjee et al., Sensors and Actuators A: Physical, Volume 114,Issues 2-3, 1 Sep. 2004, Pages 267-275.

In preferred embodiments a microneedle is only used once and thendiscarded. In some embodiments a mechanical actuator can insert andwithdraw the microneedle from the subject, discard the used needle, andreload a new microneedle. The mechanical technologies developed andmanufactured in very high volumes for very small disk drives have asimilar set of motion and low cost requirements. In preferredembodiments the actuator is a MEMS (micro machined electromechanicalsystem) device fabricated using semiconductor-like batch processes. Suchactuators include without limitation nickel titanium alloy, neumatic, orpiezo electric devices. In some embodiments the microneedles are about 1micron to about 10 microns in thickness, preferably about 2 microns toabout 6 microns in thickness, and most preferably about 4 microns inthickness. In some embodiments the microneedles are about 10 microns toabout 100 microns in height, preferably about 30 microns to about 60microns in height, and most preferably about 40 microns in height.

FIG. 1 illustrates an exemplary system of the present invention. Asillustrated, a fluidic device provides a bodily fluid from a subject andcan be inserted into a reader assembly. The fluidic device may take avariety of configurations and in some embodiments the fluidic device maybe in the form of a cartridge. An identifier (ID) detector may detect anidentifier on the fluidic device. The identifier detector communicateswith a communication assembly via a controller which transmits theidentifier to an external device. Where desired, the external devicesends a protocol stored on the external device to the communicationassembly based on the identifier. The protocol to be run on the fluidicdevice may comprise instructions to the controller of the readerassembly to perform the protocol on the fluidic device, including butnot limited to a particular assay to be run and a detection method to beperformed. Once the assay is performed on the fluidic device, a signalindicative of an analyte indicative of an influenza viral infection inthe bodily fluid sample is generated and detected by a detectionassembly. The detected signal may then be communicated to thecommunications assembly, where it can be transmitted to the externaldevice for processing, including without limitation, calculation of theanalyte concentration in the sample or determination of the presence ofthe analyte.

FIG. 2 illustrates exemplary layers of a fluidic device according to thepresent invention prior to assembly of the fluidic device which isdisclosed in more detail below. FIGS. 3 and 4 show a top and bottomview, respectively, of an exemplary fluidic device after the device hasbeen assembled. The different layers are designed and assembled to forma three dimensional fluidic channel network. A sample collection unit 4provides a sample of bodily fluid from a subject. As will be explainedin further detail below a reader assembly comprises actuating elements(not shown) can actuate the fluidic device to start and direct the flowof a bodily fluid sample and assay reagents in the fluidic device. Insome embodiments actuating elements first cause the flow of sample inthe fluidic device 2 from sample collection unit 4 to reaction sites 6,move the sample upward in the fluidic device from point G′ to point G,and then to waste chamber 8. The actuating elements then initiate theflow of reagents from reagent chambers 10 to point B′, point C′, andpoint D′, then upward to points B, C, and D, respectively, then to pointA, down to point A′, and then to waste chamber 8 in the same manner asthe sample.

A sample collection unit 4 in a fluidic device 2 may provide a bodilyfluid sample from a subject by any of the methods described above. Ifnecessary, the sample may first be processed by diluting the bodilyfluid in a dilution chamber, and or may be filtered by separating theplasma from the red blood cells in a filtration chamber. In someembodiments the sample collection unit, diluting chamber, and filtrationchamber may be the same component, and in some embodiments they may bedifferent components, or any two may be the same component and the othermay be a separate component. In some embodiments there may be more thanone sample collection unit in the fluidic device.

In some embodiments it may be desirable to detect the presence ofanalytes on a cell or viral surface, within a cell or viral membrane, orinside a cell. The difficulty of detecting such analytes is that cellsand other formed elements are particulate and components of cells do notreadily interact with traditional assay chemistries which are designedto operate on analytes in solution. Cell-surface analytes react slowlyand inefficiently with surface bound probes, and analytes inside thecell may not react at all with bound probes. To allow the detection ofsuch analytes, in some embodiments the fluidic device may include alysing assembly to lyse cells present in the bodily fluid sample. Thelysing assembly may be incorporated with the sample collection unit, adilution chamber, and/or a filtration chamber. In some embodiments thesample collection unit, dilution chamber, and lysing component arewithin the same element in the fluidic device. In some embodiments thelysing component may be incorporated with an assay reagent describedbelow.

Where desired, lysing agents may be impregnated and then dried intoporous mats, glass fiber mats, sintered frits or particles such asPorex, paper, or other similar material. Lysing agents may be dried ontoflat surfaces. Lysing agents may also be dissolved in liquid diluents orother liquid reagents. In preferred embodiments porous materials areused to store the lysing agents because they can store a lysing agent indry form likely to be very stable. They also facilitate the mixing ofthe bodily fluid sample with the lysing agent by providing a tortuouspath for the sample as it moves through the porous material. Inpreferred embodiments such porous materials have a disc shape with adiameter greater than its thickness. In some embodiments lysing agentsmay be dried onto porous materials using lyophilization, passiveevaporation, exposure to warm dry flowing gas, or other known methods.

A variety of lysing agents are available in the art and are suitable foruse in connection with the subject fluidic device. Preferred lysingagents are non-denaturing, such as non-denaturing detergents.Non-limiting examples of non-denaturing detergents include Thesit®,sodium deoxycholate, Triton®X-100, and TWEEN®20. The agents arepreferably non-volatile in embodiments where the agents are impregnatedinto a solid porous materials. In some embodiments lysing agents aremixed together. Other materials may be mixed with the lysing agents tomodify the lytic effects. Such exemplary materials may be, withoutlimitation, buffers, salts, and proteins. In preferred embodimentslysing agents will be used in amounts that are in excess of the minimumamount required to lyse cells. In some embodiments lysing agents will beused that can lyse both white and red cells.

One of the advantages of the present invention is that any reagentsnecessary to perform an assay on a fluidic device according to thepresent invention are preferably on-board, or housed within the fluidicdevice before, during, and after the assay. In this way the only inletor outlet from the fluidic device is preferably the bodily fluid sampleinitially provided by the fluidic device. This design also helps createan easily disposable fluidic device where all fluids or liquids remainin the device. The on-board design also prevents leakage from thefluidic device into the reader assembly which should remain free fromcontamination from the fluidic device.

In a preferred embodiment there is at least one reagent chamber. In someembodiments there may be two, three, four, five, six, or more, or anynumber of reagent chambers as are necessary to fulfill the purposes ofthe invention. A reagent chamber is preferably in fluid communicationwith at least one reaction site, and when the fluidic device is actuatedas described herein, reagents contained in said reagent chambers arereleased into the fluidic channels within the fluidic device.

Reagents according to the present invention include without limitationwash buffers, enzyme substrates, dilution buffers, conjugates,enzyme-labeled conjugates, sample diluents, wash solutions, samplepre-treatment reagents including additives such as detergents, polymers,chelating agents, albumin-binding reagents, enzyme inhibitors, enzymes,anticoagulants, red-cell agglutinating agents, antibodies, or othermaterials necessary to run an assay on a fluidic device. An enzymeconjugate can be either a polyclonal antibody or monoclonal antibodylabeled with an enzyme that can yield a detectable signal upon reactionwith an appropriate substrate. Non-limiting examples of such enzymes arealkaline phosphatase and horseradish peroxidase. In some embodiments thereagents comprise immunoassay reagents.

In some embodiments a reagent chamber contains approximately about 50 μlto about 1 ml of fluid. In some embodiments the chamber may containabout 100 μl of fluid. The volume of liquid in a reagent chamber mayvary depending on the type of assay being run or the sample of bodilyfluid provided. In some embodiments the reagents are initially storeddry and liquefied (e.g., dissolved or melted) upon initiation of theassay being run on the fluidic device.

FIGS. 5 and 6 illustrate an exemplary embodiment of a sealed reagentchamber. FIG. 5 shows a top, side, and bottom view of a reagent chamber.A top layer 11 contains a plurality of blisters or pouches 13. A bottomlayer 15 has a bottom surface that is bonded to the fluidic device base17 as shown in FIG. 6. The bottom layer 15 has a plurality of fluidicchannels 19 dispersed through the entire surface, where each channeltraverses the bottom layer 15. The fluid in the reagent chamber iscontained within the chamber by pressure burstable seal 21 between thefluidic channel 19 and the chamber 13. The burstable seal 21 is designedsuch that at a pre-determined pressure the seal bursts allowing thefluid in the chamber 13 to flow out into a fluidic channel 19.

FIG. 7 shows an exemplary process of filling the reagent chambers 13with, for example, reagents. Reagent chambers 13 may be filled withfluid using a fill channel and a vacuum draw channel. The process offilling the reagents involves first removing all the air from thechamber. This is done by drawing a vacuum through the vacuum drawchannel. Once the vacuum is drawn, a permanent seal is placed betweenthe fill channel and the vacuum draw channel. Next, required reagentsare dispensed into the chamber through the fill channel. Then, apermanent seal is placed between the chamber and the fill channel. Thisensures that when the chamber is compressed, the fluid can flow in onlyone direction, towards the burstable seal. If the compression imparts apressure larger than the burst pressure of seal, the seal bursts and thefluid flows into the fluidic channel.

FIGS. 8 and 9 illustrate an embodiment of a fluidic device in operationwith actuating elements as described herein. Fluidic device 2 contains areagent chamber 10 and a layer of burstable foil 12 enclosing thereagent chamber. Above the burstable foil 12 is a portion of themicrofluidic circuit 14. A tough, but elastomeric top cover 16 acts asthe top layer of the fluidic device 2. The reader assembly includes avalve actuation plate 18. Securely attached to the plate 18 is anon-coring needle 20 such that when the plate is lowered, the sharp edgeof the needle contacts the elastomeric cover 16. The top cover couldalso be made of flexible silicone material that would act as a moistureimpermeable seal. This embodiment also provides a solution to liquidevaporation and leakage from a fluidic device by isolating any liquidreagents in the fluidic device from any dry reagents until the assay isinitiated.

In preferred embodiments the reagent chamber and sample collection unitare fluidly connected to reaction sites where bound probes can detect ananalyte of interest in the bodily fluid sample using the assay. Areaction site could then provide a signal indicative of the presence ofthe analyte of interest, which can then be detected by a detectiondevice described in detail herein below.

In some embodiments the reactions sites are flat but they may take on avariety of alternative surface configurations. The reaction sitepreferably forms a rigid support on which a reactant can be immobilized.The reaction site surface is also chosen to provide appropriatelight-absorbing characteristics. For instance, the reaction site may befunctionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon,or any one of a wide variety of gels or polymers such as(poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,polycarbonate, polypropylene, or combinations thereof. Other appropriatematerials may be used in accordance with the present invention.

A reactant immobilized at a reaction site can be anything useful fordetecting an analyte of interest in a sample of bodily fluid. Forinstance, such reactants include without limitation, antibodies, cellmembrane receptors, monoclonal antibodies and antisera reactive with aspecific analyte indicative of an influenza viral infection. Variouscommercially available reactants such as a host of polyclonal andmonoclonal antibodies specifically developed for specific analytes canbe used.

A preferred class of reactants are antibodies. As used herein, an“antibody” (interchangeably used in plural form) is an immunoglobulinmolecule capable of specific binding to a target, such as an analyte ina bodily fluid, through at least one antigen recognition site, locatedin the variable region of the immunoglobulin molecule. As used herein,the term encompasses not only intact antibodies, but also fragmentsthereof (such as Fab, Fab′, F(ab′)₂, Fv, single chain (ScFv), mutantsthereof, fusion proteins, humanized antibodies, and any other modifiedconfiguration of the immunoglobulin molecule that comprises an antigenrecognition site of the required specificity.

The subject methods and apparatus can utilize antibody reactants thatare commercially available or generated de novo. Laboratory methods forproducing polyclonal antibodies and monoclonal antibodies, are known inthe art. For example, see Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York (1988) and Sambrook etal. (1989). Briefly, monoclonal antibodies useful for the presentinvention can be biologically produced by introducing an antigen of aninfluenza virus into an animal, e.g., mouse or rat. The antibodyproducing cells in the animal are isolated and fused with myeloma cellsor heteromyeloma cells to produce hybrid cells or hybridomas.

Particular isotypes of a monoclonal antibody can be prepared eitherdirectly by selecting from the initial fusion, or prepared secondarily,from a parental hybridoma secreting a monoclonal antibody of differentisotype by using the sib selection technique to isolate class switchvariants using the procedure described in Steplewski et al. (1985) Proc.Natl. Acad. Sci. 82:8653 or Spira et al. (1984) J. Immunol. Methods74:307.

The antibody reactants can be linked (i.e., conjugated) to a suitabledetectable label depending on the particular assay reaction.

In some embodiments a reactant detects an analyte indicative of aninfluenza type A, type B, or type C viral infection. The analyte maycomprise at least one surface glycoprotein of an influenza virus.Exemplary surface glycoproteins are, without limitation, a hemagglutininand a neuraminidase. Hemagglutinin surface proteins include H1, H2, H3,H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16.Neuraminidase surface proteins include N1, N2, N3, N4, and N5.

In some embodiments the reactants detect a plurality of analytes, atleast two of which are indicative of an influenza viral infection in asample of bodily fluid. The analytes may be indicative of an influenzatype A, type B, or type C viral infection. The analytes may comprise aplurality of surface glycoproteins of an influenza virus. In someembodiments the plurality of surface glycoproteins comprises ahemagglutinin and a neuraminidase. The hemagglutinin may be selectedfrom the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10,H11, H12, H13, H14, H15, and H16, and the neuraminidase may be selectedfrom the group consisting of N1, N2, N3, N4, and N5. In preferredembodiments the hemagglutinin is H5 and the neuraminidase is N1.

One skilled in the art will appreciate that there are many ways ofimmobilizing various reactants onto a support where reaction can takeplace. The immobilization may be covalent or noncovalent, via a linkermoiety, or tethering them to an immobilized moiety. These methods arewell known in the field of solid phase synthesis and micro-arrays (Beieret al., Nucleic Acids Res. 27:1970-1-977 (1999). Non-limiting exemplarybinding moieties for attaching either nucleic acids or proteinaceousmolecules such as antibodies to a solid support include streptavidin oravidin/biotin linkages, carbamate linkages, ester linkages, amide,thiolester, (N)-functionalized thiourea, functionalized maleimide,amino, disulfide, amide, hydrazone linkages, and among others. Inaddition, a silyl moiety can be attached to a nucleic acid directly to asubstrate such as glass using methods known in the art.

In some embodiments there are more than one reaction sites which canallow for detection of multiple analytes of interest from the samesample of bodily fluid. In some embodiments there are 2, 3, 4, 5, 6, ormore reaction sites, or any other number of reaction sites as may benecessary to carry out the intent of the invention.

In embodiments with multiple reaction sites on a fluidic device, eachreaction site may be immobilized with a reactant different from areactant on a different reaction site. In a fluidic device with, forexample, three reaction sites, there may be three different probes, eachbound to a different reaction site to bind to three different analytesof interest in the sample. In some embodiments there may be differentreactants bound to a single reaction site if, for example, a CCD withmultiple detection areas were used as the detection device, such thatmultiple different analytes could be detected in a single reaction site.The capability to use multiple reaction sites in addition to multipledifferent probes on each reaction site enables the high-throughputcharacteristics of the present invention.

In preferred embodiments of the invention the fluidic device includes atleast one waste chamber to trap or capture all liquids after they havebeen used in the assay. In preferred embodiments, there is more than onewaste chamber, at least one of which is to be used with a calibrationassembly described herein below. On-board waste chambers also allow thedevice to be easily disposable. The waste chamber is preferably influidic communication with at least one reaction site.

At least one of these channels will typically have small cross sectionaldimensions. In some embodiments the dimensions are from about 0.01 mm toabout 5 mm, preferably from about 0.03 mm to about 3 mm, and morepreferably from about 0.05 mm to about 2 mm. Fluidic channels in thefluidic device may be created by, for example without limitation,precision injection molding, laser etching, or any other technique knownin the art to carry out the intent of the invention.

To ensure that a given assay response (e.g. a photon count) produced ata reaction site correlates with an accurate concentration of an analyteof interest in a sample, it is preferably advantageous to calibrate thefluidic device before detecting the response (e.g., detecting photons).Calibrating a fluidic device at the point of manufacturing for examplemay be insufficient to ensure an accurate analyte concentration isdetermined because a fluidic device may be shipped prior to use and mayundergo changes in temperature, for example, so that a calibrationperformed at manufacturing does not take into effect any subsequentchanges to the structure of the fluidic device or reagents containedtherein. In a preferred embodiment of the present invention, a fluidicdevice has a calibration assembly that mimics the assay assembly incomponents and design except that a sample is not introduced into thecalibration assembly. Referring to FIGS. 3 and 4, a calibration assemblyoccupies about half of the fluidic device 2 and includes reagentchambers 32, reactions sites 34, a waste chamber 36, and fluidicchannels 38. Similar to the assay assembly, the number of reagentchambers and reaction sites may vary depending on the assay being run onthe fluidic device and the number of analytes being detected.

Where desired, a sensor for assessing the reliability of an assay for ananalyte in a bodily fluid with the use of the subject fluidic device canbe provided together with the fluidic device, the reader and/or withinthe packaging of the subject system. The sensor is capable of detectinga change in operation parameters under which the subject system normallyoperates. The operation parameters include but are not limited totemperature, humidity, and pressure, which may affect the performance ofthe present system.

A fluidic device and reader assembly may, after manufacturing, beshipped to the end user, together or individually. As a reader assemblyis repeatedly used with multiple fluidic devices, it may be necessary tohave sensors on both the fluidic device and reader assembly to detectsuch changes during shipping, for example. During shipping, pressure ortemperature changes can impact the performance of a number of componentsof the present system, and as such a sensor located on either thefluidic device or reader assembly can relay these changes to, forexample, the external device so that adjustments can be made duringcalibration or during data processing on the external device. Forexample, if the pressure or temperature of a fluidic device reached acertain level during shipping, a sensor located on the fluidic devicecould detect this change had occurred and convey this information to thereader assembly when it is inserted into the reader assembly by theuser. There may be an additional detection device in the reader assemblyto perform this, or such a device may be incorporated into anothersystem component. In some embodiments this information may be wirelesslytransmitted to either the reader assembly or the external device.Likewise, a sensor in the reader assembly can detect similar changes. Insome embodiments, it may be desirable to have a sensor in the shippingpackaging as well, either instead of in the system components or inaddition thereto.

Manufacturing of the fluidic channels may generally be carried out byany number of microfabrication techniques that are well known in theart. For example, lithographic techniques are optionally employed infabricating, for example, glass, quartz or silicon substrates, usingmethods well known in the semiconductor manufacturing industries such asphotolithographic etching, plasma etching or wet chemical etching.Alternatively, micromachining methods such as laser drilling,micromilling and the like are optionally employed. Similarly, forpolymeric substrates, well known manufacturing techniques may also beused. These techniques include injection molding or stamp moldingmethods where large numbers of substrates are optionally produced using,for example, rolling stamps to produce large sheets of microscalesubstrates or polymer microcasting techniques where the substrate ispolymerized within a micromachined mold.

In some embodiments at least one of the different layers of the fluidicdevice may be constructed of polymeric substrates. Non limiting examplesof polymeric materials include polystyrene, polycarbonate,polypropylene, polydimethysiloxanes (PDMS), polyurethane,polyvinylchloride (PVC), and polysulfone.

The fluidic device may be manufactured by stamping, thermal bonding,adhesives or, in the case of certain substrates, for example, glass, orsemi-rigid and non-rigid polymeric substrates, a natural adhesionbetween the two components. In some embodiments the fluidic device ismanufactured by ultrasonic or acoustic welding.

FIG. 2 shows one embodiment of the invention in which fluidic device 2is comprised of 7 layers. Features as shown are, for example, cut in thepolymeric substrate such that when the layers are properly positionedwhen assembly will form a fluidic network. In some embodiments more orfewer layers may be used to construct a fluidic device to carry out thepurpose of the invention.

One objective of the present invention is to prevent fluid inside afluidic device from contacting the components of a reader assembly whichmay need to remain dry and or uncontaminated, and also to preventcontamination to a detection device within the reader assembly. A leakin the fluidic device could result in liquids, for example reagents orwaste, escaping from the fluidic device and contaminating the reader. Inother embodiments a liquid absorbing material, such as polymericmaterials found in diapers, could be placed within a portion of thefluidic channel or waste chamber to absorb the waste liquid. Anon-limiting example of such a polymer is sodium polyacrylate. Suchpolymers can absorb fluids hundreds of times their weight. Hence, onlyminute quantities of such polymeric materials may be required toaccomplish the goal of absorbing leaked fluids. In some embodiments awaste chamber is filled with a superabsorbent material. In someembodiments leaked liquid may be converted into a gel or other solid orsemi-solid form.

Another objective of the present system is to provide a fluidic devicethat can run a variety of assays on a fluidic device. A protocoldependent on the identity of the fluidic device may be transferred froman external device where it can be stored to a reader assembly to enablethe reader assembly to carry out the specific protocol on the fluidicdevice. In preferred embodiments, the fluidic device has an identifier(ID) that is detected or read by an identifier detector describedherein. The identifier can then be communicated to a communicationassembly, where it can then be transferred or transmitted to an externaldevice.

In some embodiments the identifier may be a bar code identifier with aseries of black and white lines, which can be read by an identifierdetector such as a bar code reader, which are well known. Otheridentifiers could be a series of alphanumerical values, colors, raisedbumps, or any other identifier which can be located on a fluidic deviceand be detected or read by an identifier detector. In some embodimentsthe identifier may comprise a storage or memory device and can transmitinformation to an identification detector. In some embodiments bothtechniques may be used.

Once a bodily fluid sample is provided to a fluidic device, it isinserted in a reader assembly. In some embodiments the fluidic device ispartially inserted manually, and then a mechanical switch in the readerassembly automatically properly positions the fluidic device inside thereader assembly. Any other mechanism known in the art for inserting adisk or cartridge into a device may be used as well. In some embodimentsonly manual insertion may be required.

In some embodiments the reader assembly comprises an identifier detectorfor detecting or reading an identifier on the fluidic device, acontroller for automatically controlling the detection assembly and alsomechanical components of the reader assembly, for example, pumps and/orvalves for controlling or directing fluid through the fluidic device, adetection device for detecting a signal created by an assay run on thefluidic device, and a communication assembly for communicating with anexternal device.

An identifier detector detects an identifier on the fluidic device whichis communicated to a communication assembly. In some embodiments theidentifier detector can be a bar code scanner-like device, reading a barcode on a fluidic device. The identifier detector may also be an LEDthat emits light which can interact with an identifier which reflectslight and is measured by the identifier detector to determine theidentity of a fluidic device.

In preferred embodiments the reader assembly houses a controller whichcontrols a pump and a series of valves to control and direct the flow ofliquid within the fluidic device. In some embodiments the readerassembly may comprises multiple pumps. The sample and reagents arepreferably pulled through the fluidic channels by a vacuum force createdby sequentially opening and closing at least one valve while activatinga pump within the reader assembly. Methods of using at least one valveand at least one pump to create a vacuum force are well known. While anegative pulling force may be used, a positive pushing force may also begenerated by at least one pump and valve according to the presentinvention. In other embodiments movement of fluid on the fluidic devicemay be by electro-osmotic, capillary, piezoelectric, or microactuatoraction.

FIGS. 8 and 9 illustrate an exemplary sequence to initiate the flow of areagent within the fluidic device. An actuation plate 18 in the readerassembly comprises a non-coring needle or pin 20 which when loweredflexes the top cover 16, as it is preferably made of strong, flexibleelastomeric material. However, the easily rupturable foil 12 thenruptures due to the stress induced by the flexing of top cover 16.Valves located downstream to the reagent chamber puncture differentareas of foil in the fluidic device and can then work in tandem with apump within the reader assembly to create a vacuum force to pull thereagent out of the reagent chamber 6 into a fluidic channel and thendirect the flow of the reagent to a reaction site. At least one valve ispreferably fluidically connected to a pump housed within the readerassembly. The non-coring needle or pin 20 is removed from the fluidicdevice when the device is removed from the reader assembly. One of theadvantages of this embodiment is that no on-chip pump is required,which, at least, decreases the size and cost of the fluidic device, andallows the device to be disposable.

A reaction assembly preferably houses a detection assembly for detectinga signal produced by at least one assay on the fluidic device. FIG. 1illustrates an exemplary position of a detection device of the presentinvention in relation to the fluidic device which is below the fluidicdevice. The detection assembly may be above the fluidic device or at adifferent orientation in relation to the fluidic device based on, forexample, the type of assay being performed and the detection mechanismbeing employed.

In preferred embodiments an optical detector is used as the detectiondevice. Non-limiting examples include a photodiode, photomultiplier tube(PMT), photon counting detector, or charge-coupled device (CCD). In someembodiments a pin diode may be used. In some embodiments a pin diode canbe coupled to an amplifier to create a detection device with asensitivity comparable to a PMT. Some assays may generate luminescenceas described herein. In some embodiments chemiluminescence is detected.In some embodiments a detection assembly could include a plurality offiber optic cables connected as a bundle to a CCD detector or to a PMTarray. The fiber optic bundle could be constructed of discrete fibers orof many small fibers fused together to form a solid bundle. Such solidbundles are commercially available and easily interfaced to CCDdetectors.

In some embodiments, the detection system may comprise non-opticaldetectors or sensors for detecting a particular parameter of a subject.Such sensors may include temperature, conductivity, potentiometric, andamperometric, for compounds that are oxidized or reduced, for example,O₂, H₂O₂, and I₂, or oxidizable/reducible organic compounds.

A communication assembly is preferably housed within the reader assemblyand is capable of transmitting and receiving information wirelessly froman external device. Such wireless communication may be bluetooth or RTMtechnology. Various communication methods can be utilized, such as adial-up wired connection with a modem, a direct link such as a T1, ISDN,or cable line. In preferred embodiments a wireless connection isestablished using exemplary wireless networks such as cellular,satellite, or pager networks, GPRS, or a local data transport systemsuch as Ethernet or token ring over a local area network. In someembodiments the information is encrypted before it is transmitted over awireless network. In some embodiments the communication assembly maycontain a wireless infrared communication component for sending andreceiving information.

In some embodiments the communication assembly can have a memory orstorage device, for example localized RAM, in which the informationcollected can be stored. A storage device may be required if informationcan not be transmitted at a given time due to, for example, a temporaryinability to wirelessly connect to a network. The information can beassociated with the fluidic device identifier in the storage device. Insome embodiments the communication assembly can retry sending the storedinformation after a certain amount of time. In some embodiments thememory device can store the information for a period of ten days beforeit is erased.

In preferred embodiments an external device communicates with thecommunication assembly within the reader's assembly. An external devicecan wirelessly communicate with a reader assembly, but can alsocommunicate with a third party, including without limitation a patient,medical personnel, clinicians, laboratory personnel, or others in thehealth care industry.

In some embodiments the external device can be a computer system,server, or other electronic device capable of storing information orprocessing information. In some embodiments the external device includesone or more computer systems, servers, or other electronic devicescapable of storing information or processing information. In someembodiments an external device may include a database of subjectinformation, for example but not limited to, medical records or subjecthistory, clinical trial records, or preclinical trial records. Inpreferred embodiments, an external device stores protocols to be run ona fluidic device which can be transmitted to the communication assemblyof a reader assembly when it has received an identifier indicating whichfluidic device has been inserted in the reader assembly. In someembodiments a protocol can be dependent on a fluidic device identifier.In some embodiments the external device stores more than one protocolfor each fluidic device. In other embodiments subject information on theexternal device includes more than one protocol. In preferredembodiments the external server stores mathematical algorithms toprocess a photon count sent from a communication assembly and in someembodiments to calculate the analyte concentration in a bodily fluidsample.

In some embodiments the external device can include one or more serversas are known in the art and commercially available. Such servers canprovide load balancing, task management, and backup capacity in theevent of failure of one or more of the servers or other components ofthe external device, to improve the availability of the server. A servercan also be implemented on a distributed network of storage andprocessor units, as known in the art, wherein the data processingaccording to the present invention reside on workstations such ascomputers, thereby eliminating the need for a server.

A server can includes a database and system processes. A database canreside within the server, or it can reside on another server system thatis accessible to the server. As the information in a database maycontains sensitive information, a security system can be implementedthat prevents unauthorized users from gaining access to the database.

One advantage of the present invention is that information can betransmitted from the external device back to not only the readerassembly, but to other parties or other external devices, for examplewithout limitation, a PDA or cell phone. Such communication can beaccomplished via a wireless network as disclosed herein. In someembodiments a calculated analyte concentration or other subjectinformation can be sent to, for example but not limited to, medicalpersonal or the subject.

Methods of Use

The subject apparatus and systems provide an effective means forreal-time detection of analytes indicative of an influenza viralinfection present in a bodily fluid from a subject.

One aspect of the present invention is a method of detecting an analyteindicative of an influenza viral infection in a sample of bodily fluid.The analyte may be indicative of an influenza type A, type B, or type Cviral infection. The analyte may comprise at least one surfaceglycoprotein of an influenza virus. Exemplary surface glycoproteins are,without limitation, a hemagglutinin and a neuraminidase. Hemagglutininsurface proteins include H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11,H12, H13, H14, H15, and H16. Neuraminidase surface proteins include N1,N2, N3, N4, and N5. The analyte may also comprise an antibody to asurface glycoprotein of an influenza virus.

One aspect of the present invention is a method for detecting aplurality of analytes, at least two of which are indicative of aninfluenza viral infection in a sample of bodily fluid. The analytes maybe indicative of an influenza type A, type B, or type C viral infection.The analytes may comprise a plurality of surface glycoproteins of aninfluenza virus. In some embodiments the plurality of surfaceglycoproteins comprises a hemagglutinin and a neuraminidase. Thehemagglutinin may be selected from the group consisting of H1, H2, H3,H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16, and theneuraminidase may be selected from the group consisting of N1, N2, N3,N4, and N5. In preferred embodiments the method detects for bothhemagglutinin H5 and neuraminidase N1. In one embodiment the methodprovides for detection of H5 and N1 in the same viral particle(s) (seeFIG. 15).

One further aspect of the present invention is a method for detecting aplurality of analytes incorporated into a single entity such as a viralparticle or cell or cell fragment. In this aspect the plurality ofanalytes are preferably a combination or complex of analytes, at leasttwo of which are indicative of an influenza viral infection in a sampleof bodily fluid. The analytes may be indicative of an influenza type A,type B, or type C viral infection. The plurality of analytes maycomprise a combination or complex of surface glycoproteins of aninfluenza virus. In some embodiments the plurality of analytes may be acombination of surface glycoproteins comprising a combination of ahemagglutinin and a neuraminidase. The hemagglutinin may be selectedfrom the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10,H11, H12, H13, H14, H15, and H16, and the neuraminidase may be selectedfrom the group consisting of N1, N2, N3, N4, and N5. In preferredembodiments the combination of analytes is associated with a virulentstrain of influenza such as the H5N1 combination. This aspect of theinvention is specific for detecting the combination of the plurality ofanalytes. It can distinguish between infection with a virulent strainsuch as a combination of H5N1 and a putative-infection with a differentcombination of analytes.

In some embodiments the method detects a plurality of human antibodiesto viral antigens such as antibodies to surface glycoproteins of aninfluenza virus.

In some embodiments the analyte of interest may be a complex of ananalyte indicative of an influenza viral infection in a sample of bodilyfluid and a human antibody to the analyte. The analyte may be anyanalyte indicative of an influenza viral infection described herein, butis preferably the H5 hemagglutinin, the N1 neuraminidase, or the H5N1complex of the H5 and N1 surface glycoproteins.

A further aspect of the present invention is a method for detecting aplurality of analytes, wherein at least one analyte is indicative of aninfluenza viral infection in a sample of bodily fluid, and wherein atleast one analyte is a biomarker in the sample of bodily fluidindicative of the stress imposed on the human body by the viralinfection or an indicator of the body's response to the infection. Theat least one analyte indicative of an influenza viral infection may beany analyte indicative of an influenza viral infection described herein.Exemplary biomarkers indicative of the stress imposed on the human bodyby the viral infection include, without limitation, CRP, TNFinterleukins and the like. Exemplary biomarkers indicative of the body'sdefensive reaction to the virus include antibodies to the virus,particularly of the IgM isotype.

The subject apparatus and systems have a spectrum of utility in, forexample, disease diagnosis and disease detection.

Accordingly, in one embodiment, the present invention provides a methodof detecting an analyte indicative of an influenza viral infection in abodily fluid from a subject comprises providing a fluidic devicecomprising at least one sample collection unit, an immunoassay assemblycontaining immunoassay reagents, a plurality of channels in fluidcommunication with said sample collection unit and/or said immunoassayassembly; actuating said fluidic device and directing said immunoassayreagents within said fluidic device; allowing a sample of bodily fluidsuspected to contain said analyte to react with said immunoassayreagents contained within said assay immunoassay assembly to yield adetectable signal indicative of the presence of said analyte in saidbodily fluid; and detecting said detectable signal generated from saidanalyte initially collected in said sample of bodily fluid. Preferably,a sample of bodily fluid of less than about 500 ul is used for one ormore of these applications.

As used herein, the term “subject” and “patient” is usedinterchangeably, which refers to an animal, preferably an avian (bird)or a mammalian species (for example, human). The term avian as usedherein includes poultry. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.

As used herein, in some aspects the terms “reagents” and “reactants” areused interchangeably.

In some embodiments a sample of bodily fluid can first be provided tothe fluidic device by any of the methods described herein. The fluidicdevice can then be inserted into the reader assembly. An identificationdetector housed within the reader assembly can detect an identifier ofthe fluidic device and communicate the identifier to a communicationassembly, which is preferably housed within the reader assembly. Thecommunication assembly then transmits the identifier to an externaldevice which transmits a protocol to run on the fluidic device based onthe identifier to the communication assembly. A controller preferablyhoused within the reader assembly controls actuating elements includingat least one pump and one valve which interact with the fluidic deviceto control and direct fluid movement within the device. In someembodiments the first step of the assay is a wash cycle where all thesurfaces within the fluidic device are wetted using a wash buffer. Thefluidic device is then calibrated using a calibration assembly byrunning the same reagents as will be used in the assay through thecalibration reaction sites, and then a luminescence signal from thereactions sites is detected by the detection means, and the signal isused in calibrating the fluidic device. The sample containing theanalyte is introduced into the fluidic channel. The sample may bediluted and further separated into plasma or other desired component ata filter. The separated sample now flows through the reaction sites andanalytes present therein will bind to reactants bound thereon. Theplasma of sample fluid is then flushed out of the reaction wells into awaste chamber. Depending on the assay being run, appropriate reagentsare directed through the reaction sites to carry out the assay. All thewash buffers and other reagents used in the various steps, including thecalibration step, are collected in wash tanks. The signal produced inthe reaction sites is then detected by any of the methods describedherein.

A variety of assays may be performed on a fluidic device according tothe present invention to detect an analyte of interest in a sample. Awide diversity of labels is available in the art that can be employedfor conducting the subject assays. In some embodiments labels aredetectable by spectroscopic, photochemical, biochemical, immunochemical,or chemical means. For example, useful nucleic acid labels include 32P,35S, fluorescent dyes, electron-dense reagents, enzymes, biotin,digoxigenin, or haptens and proteins for which antisera or monoclonalantibodies are available. A wide variety of labels suitable for labelingbiological components are known and are reported extensively in both thescientific and patent literature, and are generally applicable to thepresent invention for the labeling of biological components. Suitablelabels include radionucleides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties,bioluminescent labels, calorimetric labels, or magnetic particles.Labeling agents optionally include, for example, monoclonal antibodies,polyclonal antibodies, proteins, or other polymers such as affinitymatrices, carbohydrates or lipids. Detection proceeds by any of avariety of known methods, including spectrophotometric or opticaltracking of radioactive or fluorescent markers, or other methods whichtrack a molecule based upon size, charge or affinity. A detectablemoiety can be of any material having a detectable physical or chemicalproperty. Such detectable labels have been well-developed in the fieldof gel electrophoresis, column chromatograpy, solid substrates,spectroscopic techniques, and the like, and in general, labels useful insuch methods can be applied to the present invention. Thus, a labelincludes without limitation any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical, thermalor chemical means.

In some embodiments the label is coupled directly or indirectly to amolecule to be detected such as a product, substrate, or enzyme,according to methods well known in the art. As indicated above, a widevariety of labels are used, with the choice of label depending on thesensitivity required, ease of conjugation of the compound, stabilityrequirements, available instrumentation, and disposal provisions.Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule is covalently bound to a polymer. The ligand thenbinds to an anti-ligand molecule which is either inherently detectableor covalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. A number ofligands and anti-ligands can be used. Where a ligand has a naturalanti-ligand, for example, biotin, thyroxine, and cortisol, it can beused in conjunction with labeled, anti-ligands. Alternatively, anyhaptenic or antigenic compound can be used in combination with anantibody.

In some embodiments the label can also be conjugated directly to signalgenerating compounds, for example, by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,and umbelliferone. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, such as luminol and dioxetanes

Methods of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence by, for example,microscopy, visual inspection, via photographic film, by the use ofelectronic detectors such as digital cameras, charge coupled devices(CCDs) or photomultipliers and phototubes, or other detection device.Similarly, enzymatic labels are detected by providing appropriatesubstrates for the enzyme and detecting the resulting reaction product.Finally, simple colorimetric labels are often detected simply byobserving the color associated with the label. For example, conjugatedgold often appears pink, while various conjugated beads appear the colorof the bead.

In some embodiments the detectable signal may be provided byluminescence sources. “Luminescence” is the term commonly used to referto the emission of light from a substance for any reason other than arise in its temperature. In general, atoms or molecules emit photons ofelectromagnetic energy (e.g., light) when they move from an “excitedstate” to a lower energy state (usually the ground state). There aremany causes of excitation. If exciting cause is a photon, theluminescence process is referred to as “photoluminescence”. If theexciting cause is an electron, the luminescence process is referred toas “electroluminescence”. More specifically, electroluminescence resultsfrom the direct injection and removal of electrons to form anelectron-hole pair, and subsequent recombination of the electron-holepair to emit a photon. Luminescence which results from a chemicalreaction is usually referred to as “chemiluminescence”. Luminescenceproduced by a living organism is usually referred to as“bioluminescence”. If photoluminescence is the result of a spin-allowedtransition (e.g., a single-singlet transition, triplet-triplettransition), the photoluminescence process is usually referred to as“fluorescence”. Typically, fluorescence emissions do not persist afterthe exciting cause is removed as a result of short-lived excited stateswhich may rapidly relax through such spin-allowed transitions. Ifphotoluminescence is the result of a spin-forbidden transition (e.g., atriplet-singlet transition), the photoluminescence process is usuallyreferred to as “phosphorescence”. Typically, phosphorescence emissionspersist long after the exciting cause is removed as a result oflong-lived excited states which may relax only through suchspin-forbidden transitions. A “luminescent label” may have any one ofthe above-described properties.

Suitable chemiluminescent sources include a compound which becomeselectronically excited by a chemical reaction and may then emit lightwhich serves as the detectible signal or donates energy to a fluorescentacceptor. A diverse number of families of compounds have been found toprovide chemiluminescence under a variety or conditions. One family ofcompounds is 2,3-dihydro-1,4-phthalazinedione. A frequently usedcompound is luminol, which is a 5-amino compound. Other members of thefamily include the 5-amino-6,7,8-trimethoxy- and thedimethylamino[ca]benz analog. These compounds can be made to luminescewith alkaline hydrogen peroxide or calcium hypochlorite and base.Another family of compounds is the 2,4,5-triphenylimidazoles, withlophine as the common name for the parent product. Chemiluminescentanalogs include para-dimethylamino and -methoxy substituents.Chemiluminescence may also be obtained with oxalates, usually oxalylactive esters, for example, p-nitrophenyl and a peroxide such ashydrogen peroxide, under basic conditions. Other useful chemiluminescentcompounds that are also known include N-alkyl acridinum esters anddioxetanes. Alternatively, luciferins may be used in conjunction withluciferase or lucigenins to provide bioluminescence.

In some embodiments immunoassays are run on the fluidic device. Whilecompetitive binding assays, which are well known in the art, may be runin some embodiments, in certain embodiments a two-step method is usedwhich eliminates the need to mix a conjugate and a sample beforeexposing the mixture to an antibody, which may be desirable when verysmall volumes of sample and conjugate are used, as in the fluidic deviceof the present invention. A two-step assay has additional advantagesover the competitive binding assays when used with a fluidic device asdescribed herein. It combines the ease of use and high sensitivity of asandwich (competitive binding) immunoassay with the ability to assaysmall molecules.

In an exemplary two-step assay shown in FIG. 10, the sample containinganalyte (“Ag”) first flows over a reaction site containing antibodies(“Ab”). The antibodies bind the analyte present in the sample. After thesample passes over the surface, a solution with analyte conjugated to amarker (“labeled Ag”) at a high concentration is passed over thesurface. The conjugate saturates any of the antibodies that have not yetbound the analyte. Before equilibrium is reached and any displacement ofpre-bound unlabelled analyte occurs, the high-concentration conjugatesolution is washed off. The amount of conjugate bound to the surface isthen measured by the appropriate technique, and the detected conjugateis inversely proportional to the amount of analyte present in thesample.

An exemplary measuring technique for a two-step assay is achemiluminescence enzyme immunoassay as shown in FIG. 11. As is known inthe field, the marker can be a commercially available marker such asdioxitane-phosphate, which is not luminescent but becomes luminescentafter hydrolysis by, for example, alkaline phosphatase. An enzyme suchas alkaline phosphatase is also passed over the substrate to cause themarker to luminesce. In some embodiments the substrate solution issupplemented with enhancing agents such as, without limitation,fluorescein in mixed micelles, soluble polymers, or PVC which create amuch brighter signal than the luminophore alone. Moreover, an alkalinephosphatase conjugate with a higher turnover number than that used inthe commercial assay is employed. This allows signal generation toproceed much more rapidly and a higher overall signal is achieved. Theincreased sensitivity of the two-step chemiluminescent enzymeimmunoassay (TOSCA) is illustrated in FIG. 12. FIG. 12 shows that foranalytes in the picomolar concentration, TOSCA is able to provide a morerobust signal (higher sensitivity) than a competitive binding assay. Useof a two-step binding assay thus contributes to higher sensitivitycapabilities of the present invention.

Additionally, TOSCA is less sensitive to matrix effects than othermethodologies. This allows one to work with samples that have not beenextensively pre-processed using standard laboratory techniques such as,for example, solid phase extraction and chromatography. The ability ofTOSCA to assay less than ideal samples and maintain desired sensitivityis illustrated in FIG. 13. Compared to competitive binding assay, forall sample preparations (and dilutions), TOSCA has better sensitivitythan competitive binding.

One useful immunoassay that can be run on the fluidic device is ELISA(Enzyme-Linked ImmunoSorbent Assay). Performing an ELISA generallyinvolves at least one antibody capable of binding an antigen of interest(i.e., an analyte that is indicative of influenza viral infection). Asample containing or suspected to contain the antigen of interest isimmobilized on a support (e.g., a microtiter plate, a well or othersupport having a surface for immobilization) either non-specifically(e.g., via adsorption to the surface) or specifically (e.g., via captureby another antibody specific to the same antigen, in a “sandwich”ELISA). After the antigen is immobilized the detection antibody isadded, forming a complex with the antigen. The detection antibody can beconjugated to an enzyme, or can itself be detected by a secondaryantibody which is in turn conjugated to an enzyme. Upon addition of asubstrate for the conjugated enzyme, a detectable signal is generatedwhich indicates the presence and/or quantity of the antigen in thesample. The choice of substrates will depend on the enzyme conjugated.Suitable substrates include fluorogenic and chromogenic substrates. Oneof skill in the art would be knowledgeable as to the parameters that canbe modified to increase the signal detected as well as other variationsof ELISAs known in the art.

FIG. 14 illustrates a typical ELISA. As shown, a solid phase capturesurface can include an attached first antibody to which diluted plasma(sample) can be added. Analyte if present in the sample can bind to thefirst antibody and become immobilized. An enzyme reagent can be addedthat includes, for example, an antibody coupled or conjugated to anenzyme (e.g., alkaline phosphatase). If the antibody portion of theenzyme reagent can bind the analyte, then the enzyme reagent alsobecomes immobilized at the capture surface. Addition of a substrate forthe enzyme can result in a product producing an effect, for example,light that can be measured and plotted as shown. In this manner theamount of analyte present in a sample can be measured.

FIG. 15 illustrates an exemplary ELISA for use with the fluidic deviceof the invention. As shown, a solid phase capture surface of the devicecan include a first antibody, “solid phase antibody 1”, that is surfaceimmobilized and specific for a test antigen (e.g., antibody specific fora neuraminidase on a virus). If the test antigen is present in a testsample (e.g., blood) exposed to the solid phase antibody 1 then the testantigen can become immobilized (captured) at the capture surface.Subsequently provided is a second antibody that is specific for a secondtest antigen and includes a conjugated detectable compound, shown as“enzyme labeled antibody 2” (e.g., enzyme labeled antibody specific fora hemagglutinin on a virus), that can be added after the test sample(e.g., blood). Binding and subsequent detection of the second conjugatedantibody at the capture surface can indicate the presence of the firstand second test antigens in the test sample. In use, the first andsecond test antigens can include any of the neuraminidase orhemagglutinin types described herein.

Although different first and second antigens (and antibodies) are usedin the illustrated example, it is envisioned that a single type of testantigen could be detected using two forms of the same antibody (i.e., animmobilized solid phase form for antigen capture and an enzyme labeledform for detection).

The term “analytes” according to the present invention includes withoutlimitation drugs, prodrugs, pharmaceutical agents, drug metabolites,biomarkers such as expressed proteins and cell markers, antibodies,antigens, viruses, serum proteins, cholesterol, polysaccharides, nucleicacids, biological analytes, biomarker, gene, protein, or hormone, or anycombination thereof. At a molecular level, the analytes can bepolypeptide glycoprotein, polysaccharide, lipid, nucleic acid, and acombination thereof.

Of interest are biomarkers are associated with a particular disease orwith a specific disease stage. Such analytes include but are not limitedto those associated with autoimmune diseases.

Also of interest are analytes that are indicative of a microorganism.Exemplary microorganisms include but are not limited to bacterium,virus, fungus and protozoa.

The analyte may be indicative of an influenza type A, type B, or type Cviral infection. The analyte may comprise at least one surfaceglycoprotein of an influenza virus. Exemplary surface glycoproteins are,without limitation, a hemagglutinin and a neuraminidase. Hemagglutininsurface proteins include H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11,H12, H13, H14, H15, and H16. Neuraminidase surface proteins include N1,N2, N3, N4, and N5.

One aspect of the present invention is a system for detecting aplurality of analytes, at least two of which are indicative of aninfluenza viral infection in a sample of bodily fluid. The analytes maybe indicative of an influenza type A, type B, or type C viral infection.The analytes may comprise a plurality of surface glycoproteins of aninfluenza virus. In some embodiments the plurality of surfaceglycoproteins comprises a hemagglutinin and a neuraminidase. Thehemagglutinin may be selected from the group consisting of H1, H2, H3,H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16, and theneuraminidase may be selected from the group consisting of N1, N2, N3,N4, and N5. In preferred embodiments the hemagglutinin is H5 and theneuraminidase is N1. The system is capable of detecting and/orquantifying the analytes of particular interest.

By detecting the presence of the viral antigens or antibodies to theantigens, for example, the fluidic device can detect the presence of atype of influenza virus in the sample of bodily fluid from the subject.

Analytes that can be detected by the subject method also includeblood-born pathogens selected from a non-limiting group that consists ofStaphylococcus epidermidis, Escherichia coli, methicillin-resistantStaphylococcus aureus (MSRA), Staphylococcus aureus, Staphylococcushominis, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcuscapitis, Staphylococcus warneri, Klebsiella pneumoniae, Haemophilusinflunzae, Staphylococcus simulans, Streptococcus pneumoniae and Candidaalbicans.

Analytes that can be detected by the subject method also encompass avariety of sexually transmitted diseases selected from the following:gonorrhea (Neisseria gorrhoeae), syphilis (Treponena pallidum),Chlamydia (Chlamydia trachomatis), nongonococcal urethritis (Ureaplasmurealyticum), yeast infection (Candida albicans), chancroid (Haemophilusducreyi), trichomoniasis (Trichomonas vaginalis), genital herpes (HSVtype I & II), HIV I, HIV II and hepatitis A, B, C, G, as well ashepatitis caused by TTV.

It is envisioned that in some embodiments the present invention providesfor monitoring an infection by a pathogen either directly throughdetection of the pathogen or indirectly, for example, by detection of aanalyte associated with a pathogen (e.g., a viral antigen) or even bydetection of an antibody to a component or product associated with apathogen (e.g., an antibody to a viral antigen). It is also envisionedthat a pathogen can be indirectly detected through an immune-relatedresponse to the pathogen. Detection of the pathogen can be performed ona test sample from a subject that is asymptomatic or symptomatic for thepathogen. Detection of the pathogen can be performed on a test samplefrom a subject before, during or after infection with the pathogen. Assuch, it is envisioned that an early stage infection (e.g., in somecases an asymptomatic infection), or any later phase of infection can bemonitored for the pathogen of interest.

A wide range of pathogen concentrations in a sample from a subject canbe detected either directly or indirectly as discussed above using theinvention. The amount of pathogen present in a test sample can beexpressed in any of a number of ways well known in the art. By way ofnon-limiting examples, the number of pathogens can be expressed as viralburden (e.g., where the infection is a viral infection), infectiousunits (IU), and/or infectious units per million cells or milliliter(IUPM). In one example, it is envisioned that pathogens can be detectedin a test sample at a concentration of from 100 IU per ml of sample upto 1×10⁹ IU per ml of sample using the invention. In another examplepathogens can be detected from 100 IU per ml of sample up to 1000 IU perml of sample using the invention. In yet another example pathogens canbe detected from 1,000 IU per ml of sample up to 1×10⁶ IU per ml ofsample using the invention.

In a separate embodiment, the present invention provides a method ofmonitoring more than one pharmacological parameter useful for assessingefficacy and/or toxicity of an anti-influenza therapeutic agent. Themethod comprises subjecting a sample of bodily fluid from a subjectadministered with the anti-influenza therapeutic agent to a fluidicdevice for monitoring said more than one pharmacological parameter, saidfluidic device comprising at least one sample collection unit, and anassay assembly comprising reaction reagents; actuating said fluidicdevice and directing said immunoassay reagents within said fluidicdevice; allowing said sample of bodily fluid to react with immunoassayreagents to yield detectable signals indicative of the values of themore than one pharmacological parameter from said sample; and detectingsaid detectable signal generated from said sample of bodily fluid. Wheredesired, the method further involves repeating the steps at a timeinterval prompted by a wireless signal communicated to the subject.

For the purposes of this invention, a “therapeutic agent” is intended toinclude any substances that have therapeutic utility and/or potential.Such substances include but are not limited to biological or chemicalcompounds such as a simple or complex organic or inorganic molecules,peptides, proteins (e.g. antibodies) or a polynucleotides (e.g.anti-sense). A vast array of compounds can be synthesized, for examplepolymers, such as polypeptides and polynucleotides, and syntheticorganic compounds based on various core structures, and these are alsoincluded in the term “therapeutic agent”. In addition, various naturalsources can provide compounds for screening, such as plant or animalextracts, and the like. It should be understood, although not alwaysexplicitly stated that the agent is used alone or in combination withanother agent, having the same or different biological activity as theagents identified by the inventive screen. The agents and methods alsoare intended to be combined with other therapies.

Pharmacodynamic (PD) parameters according to the present inventioninclude without limitation physical parameters such as temperature,heart rate/pulse, blood pressure, and respiratory rate, and biomarkerssuch as proteins, cells, and cell markers. Biomarkers could beindicative of disease or could be a result of the action of a drug.Pharmacokinetic (PK) parameters according to the present inventioninclude without limitation drug and drug metabolite concentration.Identifying and quantifying the PK parameters in real time from a samplevolume is extremely desirable for proper safety and efficacy of drugs.If the drug and metabolite concentrations are outside a desired rangeand/or unexpected metabolites are generated due to an unexpectedreaction to the drug, immediate action may be necessary to ensure thesafety of the subject. Similarly, if any of the pharmacodynamic (PD)parameters fall outside the desired range during a treatment regime,immediate action may have to be taken as well.

In preferred embodiments physical parameter data is stored in orcompared to store profiles of physical parameter data in abioinformatics system which may be on an external device incorporatingpharmacogenomic and pharmacokinetic data into its models for thedetermination of toxicity and dosing. Not only does this generate datafor clinical trials years prior to current processes but also enablesthe elimination of current disparities between apparent efficacy andactual toxicity of drugs through real-time continuous monitoring. Duringthe go/no go decision process in clinical studies, large scalecomparative population studies can be conducted with the data stored onthe database. This compilation of data and real-time monitoring allowsmore subjects to enter clinical trials in a safe fashion earlier thancurrently allowed. In another embodiment biomarkers discovered in humantissue studies can be targeted by the device for improved accuracy indetermining drug pathways and efficacy in cancer studies.

In another embodiment, the present invention provides a method ofdetecting at least two distinct analytes indicative of an influenzaviral infection of different concentrations in a bodily fluid from asubject comprises providing a fluidic device comprising a samplecollection unit, an assay assembly, and a plurality of channels in fluidcommunication with said sample collection unit and/or said assayassembly; allowing a sample of bodily fluid to react with a plurality ofreactants contained in said assay assembly to yield signals indicativeof the concentrations of said at least two analytes; and detecting saidsignals that are indicative of the presence or absence of the at leasttwo distinct analytes, wherein said signals are detectable over a rangeof 3 orders of magnitude.

Currently, a need exists for the detecting more than one analyteindicative of an influenza viral infection where the analytes arepresent in widely varying concentration range, for example, one analyteis in the pg/ml concentration and another is in the ng/ml concentrationChemiluminescence-ELISA has the ability to simultaneously assay analytesthat are present in the same sample in a wide concentration range.Another advantage for being able to detect concentrations of differentanalytes present in a wide concentration range is the ability to relatethe ratios of the concentration of these analytes to safety and efficacyof multiple drugs administered to a subject. For example, unexpecteddrug-drug interactions can be a common cause of adverse drug reactions.A real-time, concurrent measurement technique for measuring differentanalytes would help avoid the potentially disastrous consequence ofadverse drug-drug interactions.

Being able to monitoring the rate of change of an analyte concentrationor PD or PK over a period of time in a single subject, or performingtrend analysis on the concentration, PD, or PK, whether they areconcentrations of drugs or their metabolites, can help preventpotentially dangerous situations. For example, if glucose were theanalyte of interest, the concentration of glucose in a sample at a giventime as well as the rate of change of the glucose concentration over agiven period of time could be highly useful in predicting and avoiding,for example, hypoglycemic events. Such trend analysis has widespreadbeneficial implications in drug dosing regimen. When multiple drugs andtheir metabolites are concerned, the ability to spot a trend and takeproactive measures is often desirable.

Accordingly, the present invention provides a method of performing atrend analysis on the concentration of an analyte indicative of aninfluenza viral infection in a subject. The method comprise a) providinga fluidic device comprising at least one sample collection unit, animmunoassay assembly containing immunoassay reagents, a plurality ofchannels in fluid communication with said sample collection unit and/orsaid immunoassay assembly; b) actuating said fluidic device anddirecting said immunoassay reagents within said fluidic device; c)allowing a sample of bodily fluid to react with said immunoassayreagents contained within said assay immunoassay assembly to yield adetectable signal indicative of the presence of said analyte in saidsample; d) detecting said detectable signal generated from said analytecollected in said sample of bodily fluid; and e) repeating steps a)through d) for a single subject over a period of time to detectconcentrations of said analyte, thereby performing said trend analysis.

In some embodiments, a method of detecting an analyte indicative of aninfluenza viral infection in a bodily fluid from a subject using anassay transmitted from an external device is provided. The methodcomprises providing a fluidic device comprising at least one samplecollection unit and an immunoassay assembly containing immunoassayreagents; detecting said fluidic device and wirelessly transmitting animmunoassay protocol to said device; allowing a sample of bodily fluidto react with immunoassay reagents to yield a detectable signalindicative of the presence of said analyte using said transmittedimmunoassay protocol; and detecting said detectable signal.

Communication between a reader assembly and an external storage deviceallows for a reader assembly of the present invention to download afluidic device-specific protocol to run on the fluidic device based onthe identity of the fluidic device. This allows a reader assembly to beused interchangeably with any appropriate fluidic device describedherein. In addition, the external device can store a plurality ofprotocols associated with a given fluidic device, and depending on, forexample, a subject's treatment regime or plan, different protocols canbe communicated from the external device to the reader assembly to berun on the fluidic device to detect a variety of analytes indicative ofan influenza viral infection. The external device can also store aplurality of protocols associated not only with a fluidic device, butalso with a particular subject or subjects, such that a protocol can beassociated with a subject as well as with a fluidic device.

The present invention allows for automatic quantification of apharmacological parameter of a subject as well as automatic comparisonof the parameter with, for example, the subject's medical records whichmay include a history of the monitored parameter, or medical records ofanother group of subjects. Coupling real-time analyte monitoring with anexternal device which can store data as well as perform any type of dataprocessing or algorithm, for example, provides a device that can assistwith typical subject care which can include, for example, comparingcurrent subject data with past subject data. The present inventiontherefore creates a business method which effectively performs at leastpart of the monitoring of a subject that is currently performed bymedical personnel.

Significant advantages are provided by the envisioned network. As allthe information is securely channeled through the internet, this allowsthe simultaneous sharing of information with various interested parties,while satisfying the appropriate clinical, regulatory and businessneeds.

In some embodiments, the present invention provides a method oftransmitting a pharmacological parameter of a subject via a handhelddevice comprises providing a fluidic device comprising at least onesample collection unit and an assay assembly; allowing a sample ofbodily fluid to react with reactants contained within said assayassembly to yield a detectable signal indicative of the presence of saidanalyte indicative of an influenza virus; detecting said detectablesignal; transmitting said signal to an external device; processing saidsignal in said external device; and transmitting said processed signalvia a handheld device.

One advantage of the current invention is that assay results can besubstantially immediately communicated to any third party that maybenefit from obtaining the results. For example, once the analyteconcentration is determined at the external device, it can betransmitted to a patient or medical personnel who may need to takefurther action. The communication step to a third party can be performedwirelessly as described herein, and by transmitting the data to a thirdparty's hand held device, the third party can be notified of the assayresults virtually anytime and anywhere. Thus, in a time-sensitivescenario, a patient may be contacted immediately anywhere if urgentmedical action may be required.

In some embodiments a method of automatically selecting a protocol to berun on a fluidic device comprises providing a fluidic device comprisingan identifier detector and an identifier; detecting said identifier withsaid identifier detector; transferring said identifier to an externaldevice; and selecting a protocol to be run on said fluidic device from aplurality of protocols on said external device associated with saididentifier.

By detecting each fluidic device based on an identifier associated withthe fluidic device after it is inserted in the reader assembly, thesystem of the present invention allows for fluidic device-specificprotocols to be downloaded from an external device and run on thefluidic device. In some embodiments the external device can store aplurality of protocols associated with the fluidic device or associatedwith a particular subject or group of subjects. For example, when theidentifier is transmitted to the external device, software on theexternal device can obtain the identifier. Once obtained, software onthe external device, such as a database, can use the identifier toidentify protocols stored in the database associated with theidentifier. If only one protocol is associated with the identifier, forexample, the database can select the protocol and software on theexternal device can then transmit the protocol to the communicationassembly on the reader assembly. The ability to use protocolsspecifically associated with a fluidic device allows for any appropriatefluidic device to be used with a single reader assembly, and thusvirtually any analyte of interest can be detected with a single readerassembly.

In some embodiments multiple protocols may be associated with a singleidentifier. For example, if it is beneficial to detect from the samesubject an analyte once a week, and another analyte twice a week,protocols on the external device associated with the identifier can alsoeach be associated with a different day of the week, so that when theidentifier is detected, the software on the external device can select aspecific protocol that is associated with the day of the week.

In some embodiments a subject may be provided with a plurality offluidic devices to use to detect a variety of analytes. A subject may,for example, use different fluidic devices on different days of theweek. In some embodiments the software on the external deviceassociating the identifier with a protocol may include a process tocompare the current day with the day the fluidic device is to be usedbased on a clinical trial for example. If for example, the two days ofthe week are not identical, the external device can wirelessly sendnotification to the subject using any of the methods described herein orknown in the art to notify them that an incorrect fluidic device is inthe reader assembly and also of the correct fluidic device to use thatday. This example is only illustrative and can easily be extended to,for example, notifying a subject that a fluidic device is not being usedat the correct time of day.

In some embodiments, the present invention provides a method ofobtaining pharmacological data useful for assessing efficacy and/ortoxicity of an anti-influenza pharmaceutical agent from a test animal.The method involves the steps of a) providing a fluidic devicecomprising at least one sample collection unit, an assay assembly; and aplurality of channels in fluid communication with said sample collectionunit and/or said assay assembly; b) allowing a sample of biologicalfluid of less than about 50 ul to react with reactants contained withinsaid assay assembly to yield a detectable signal generated from ananalyte indicative of an influenza viral infection initially collectedin said sample that is indicative of a pharmacological parameter; and c)detecting said detectable signal; and d) repeating the reaction anddetection steps with a second sample of biological fluid from the sametest animal. In a related embodiment, the present invention provides amethod comprising a) providing a fluidic device comprising at least onesample collection unit, an assay assembly; and a plurality of channelsin fluid communication with said sample collection unit and/or saidassay assembly; b) allowing a sample of biological fluid to react withreactants contained within said assay assembly to yield a detectablesignal generated from an analyte initially collected in said sample thatis indicative of a pharmacological parameter; and c) detecting saiddetectable signal; and d) repeating the reaction and detection stepswith a second sample of biological fluid from the same test animal,wherein the animal is not subjected to anesthesia.

When using laboratory animals in preclinical testing of ananti-influenza pharmaceutical agent, it is often necessary to kill thetest subject to extract enough blood to perform an assay to detect ananalyte of interest. This has both financial and ethical implications,and as such it may be advantageous to be able to draw an amount of bloodfrom a test animal such that the animal does not need to be killed. Inaddition, this can also allow the same test animal to be tested withmultiple pharmaceutical agents at different times, thus allowing for amore effective preclinical trial. On average, the total blood volume ina mouse, for example, is 6-8 mL of blood per 100 gram of body weight. Abenefit of the current invention is that only a very small volume ofblood is required to perform preclinical trials on mice or other smalllaboratory animals. In some embodiment between about 1 microliter andabout 50 microliters are drawn. In preferred embodiment between about 1microliter and 10 microliters are drawn. In preferred embodiments about5 microliters of blood are drawn.

A further advantage of keeping the test animal alive is evident in apreclinical time course study. When multiple mice, for example, are usedto monitor the levels of an analyte in a test subject's bodily fluidover time, the added variable of using multiple subjects is introducedinto the trial. When, however, a single test animal can be used as itsown control over a course of time, a more accurate and beneficialpreclinical trial can be performed.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A method of detecting an influenza viral particle in a sample ofbodily fluid using a fluidic device comprising a sample collection unit,an assay assembly, and a waste chamber, the method comprising: receivingthe sample of bodily fluid from a subject suspected to contain theinfluenza viral particle at the sample collection unit of the fluidicdevice; providing the sample to the assay assembly of the fluidic devicethrough the sample collection unit, wherein the assay assembly comprisesa first reagent chamber, a second reagent chamber, a reaction site, anda fluidic channel that connects the first and second reagent chamberswith the reaction site, and wherein the first reagent chamber comprisesa first immunoassay reagent and the second reagent chamber comprises asecond immunoassay reagent; releasing the first and second immunoassayreagents from the first and second reagent chambers to the reaction siteof the assay assembly through the fluidic channel by one or moreactuating elements; allowing the sample to react with the first andsecond immunoassay reagents at the reaction site of the assay assembly,wherein the first immunoassay reagent binds to a hemagglutinin moleculeto form a first immune complex on the influenza viral particle, whereinthe second immunoassay reagent binds to a neuraminidase molecule to forma second immune complex on the influenza viral particle, and wherein thefirst and second immune complexes yield one or more signals indicativeof the simultaneous presence of hemagglutinin and neuraminidase on theinfluenza viral particle; and detecting the influenza viral particle inthe sample based on detection of the one or more signals indicative ofthe simultaneous presence of hemagglutinin and neuraminidase.
 2. Themethod of claim 1, wherein the one or more actuating elements compriseat least one pump and one valve to control and direct movement of fluidsin the fluidic device.
 3. The method of claim 1, wherein detection ofthe one or more signals indicative of the simultaneous presence ofhemagglutinin and neuraminidase is indicative of an influenza type Aviral infection.
 4. The method of claim 1, wherein detection of the oneor more signals indicative of the simultaneous presence of hemagglutininand neuraminidase is indicative of an influenza type B viral infection.5. The method of claim 1, wherein the hemagglutinin is selected from thegroup consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12,H13, H14, H15, and H16, and the neuraminidase is selected from the groupconsisting of N1, N2, N3, N4, and N5.
 6. The method of claim 1, whereinthe hemagglutinin is H5 and the neuraminidase is N1.
 7. A system fordetecting at least one analyte indicative of influenza in a sample ofbodily fluid, the system comprising: a fluidic device comprising: asample collection unit configured to receive the sample of bodily fluid;and an assay assembly configured to yield one or more signals indicativeof the presence of the at least one analyte in the sample of bodilyfluid; an identification detector; a communication assembly; and adetection assembly, wherein the identification detector is configuredto: detect an identifier of the fluidic device; and transmit theidentifier to the communication assembly, wherein the communicationassembly is configured to: receive, from an external device, a protocolto execute on the fluidic device selected in response to detection ofthe identifier, wherein the protocol comprises instructions to run anassay on the fluidic device; and wherein the detection assembly isconfigured to: detect the at least one analyte in the sample during theassay by detecting the one or more signals based on the instructions inthe protocol received from the external device; and transmit informationregarding the detected one or more signals to the external device viathe communication assembly, where a comparison of the detected one ormore signals with a known profile associated with the at least oneanalyte is performed.
 8. The system of claim 7, wherein the at least oneanalyte comprises at least one of a hemagglutinin molecule and aneuraminidase molecule.
 9. The system of claim 8, wherein detection ofthe one or more signals indicative of the presence of the at least oneanalyte in the sample of bodily fluid comprises detection of asimultaneous presence of hemagglutinin and neuraminidase in the sampleof bodily fluid, and wherein detection of the one or more signals isindicative of an influenza type A viral infection or an influenza type Bviral infection.
 10. The system of claim 7, wherein the communicationassembly is further configured to: receive the protocol from theexternal device in response to transmitting the identifier to theexternal device.
 11. A method of detecting at least one analyteindicative of influenza in a sample of bodily fluid, the methodcomprising: receiving the sample of bodily fluid at a sample collectionunit of a fluidic device, wherein the fluidic device further comprisesan assay assembly; detecting an identifier of the fluidic device by anidentification detector; transmitting, by the identification detector,the identifier to a communication assembly; transmitting, by thecommunication assembly, the identifier to an external device; receiving,from the external device, a protocol to execute on the fluidic deviceselected in response to detection of the identifier, wherein theprotocol comprises instructions to run an assay on the fluidic device;allowing the sample of bodily fluid to react with at least one reactantimmobilized in the assay assembly of the fluidic device during theassay, wherein the assay assembly is configured to yield one or moresignals indicative of the presence of the at least one analyteindicative of influenza in the sample of bodily fluid; detecting, by adetection assembly, the at least one analyte indicative of influenza inthe sample by detecting the one or more signals based on theinstructions in the protocol received from the external device; andtransmitting, by the detection assembly, information regarding thedetected one or more signals to the external device via thecommunication assembly, where a comparison of the detected one or moresignals with a known profile associated with the at least one analyte isperformed.
 12. The method of claim 11, wherein the at least one analytecomprises at least one of a hemagglutinin molecule and a neuraminidasemolecule.
 13. The method of claim 12, wherein detecting the one or moresignals indicative of the presence of the at least one analyte in thesample of bodily fluid comprises detecting a simultaneous presence ofhemagglutinin and neuraminidase in the sample of bodily fluid.
 14. Themethod of claim 13, wherein detecting the one or more signals isindicative of an influenza type A viral infection or an influenza type Bviral infection.
 15. The method of claim 13, wherein the hemagglutininis selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8,H9, H10, H11, H12, H13, H14, H15, and H16, and the neuraminidase isselected from the group consisting of N1, N2, N3, N4, and N5.
 16. Themethod of claim 13, wherein the hemagglutinin is H5 and theneuraminidase is N1.
 17. The method of claim 11, further comprising:selecting the protocol from a plurality of protocols stored on theexternal device based on the identifier.
 18. The method of claim 11,further comprising: moving the sample of bodily fluid from the samplecollection unit to the assay assembly in the fluidic device.
 19. Themethod of claim 11, further comprising: moving the sample of bodilyfluid from the assay assembly to a waste chamber in the fluidic device.20. The method of claim 11, further comprising: processing the sample ofbodily fluid prior to receiving the sample at the sample collection unitof the fluidic device by dilution.